Novel thermostable gluconate dehydratase and use thereof

ABSTRACT

The present invention relates to a novel thermostable gluconate dehydratase from the thermoacidophilic archaeon  Sulfolobus solfataricus , a coding sequence, and an expression system. The gluconate dehydratase has a molecular weight of about 320,000 to 380,000 daltons as the native protein, and about 40,000 to 50,000 daltons as the monomer protein, and catalyzes the dehydration reaction of aldonic acids to 2-keto-3-deoxy derivatives at temperatures of less than 120° C. The gluconate dehydratase can be produced from native or recombinant host cells and thereby used in the pharmaceutical, agricultural, and other industries.

TECHNICAL FIELD

The present invention relates to a novel nucleic acid coding for athermostable gluconate dehydratase from the archaeon Sulfolobussolfataricus, a novel polypeptide coded by the nucleic acid, and usethereof, as well as a method for preparing and isolating the recombinantgluconate dehydratase, and catalyzing aldonic acids to 2-keto-3-deoxyderivatives.

BACKGROUND ART

The hyperthermophilic archaea are microorganisms that grow optimally ata temperature above 80° C. Many species of these extremely thermophilicbacteria-like organisms have been isolated, mainly from volcanically andgeothermally heated hydrothermal environments, such as solfataricfields, hot springs, and submarine hot vents.

The discovery of microorganisms growing optimally around 80° C. is ofconsiderable interest in both academic and industrial communities. Boththe organisms and their enzymes have the potential to bridge the gapbetween biochemical catalysis and many industrial chemical conversions.However, knowledge of the metabolism of the hyperthermophilicmicroorganisms is presently very limited.

In many hyperthermophilic archaea habited in these biotops, the orderSulfolobales which includes the genus Sulfolobus, have achemolithoautotrophic metabolism which converts elemental sulfur tohydrogen sulfide using organic compounds or hydrogen as an electrondonor. Although Sulfolobus is the sulfur-oxidizing genus, this genus cangrow chemoheterotrophically to a high cell density using sugars.Sulfolobus solfataricus optimally grows at 80-85° C. and pH 2-4,utilizing glucose as the sole carbon and energy source (Grogan, J.Bacteriol. 171:6710-6719, 1989)). In Sulfolobus, the glucose metabolismpathway was first analyzed with ¹⁴C-glucose-label experiments by De Rosaet al. (Biochem. J. 224: 407-414, 1984). De Rosa's experiment shows thatSulfolobus can convert glucose to pyruvate through a modifiedEntner-Doudoroff (ED) pathway which produces non-phosphorylatedintermediates such as gluconate, 2-keto-3-deoxygluconate (KDG), andglyceraldehyde. The first reaction of the non-phosphorylated ED pathwayin S. solfatarcus involves the NAD(P)⁺-dependent oxidation of glucose togluconate, catalyzed by glucose dehydrogenase. Gluconate is thendehydrated by gluconate dehydratase (EC 4.2.1.39) to2-keto-3-deoxygluconate (KDG), which is cleaved to pyruvate andglyceraldehydes, and catalyzed by KDG-alolase (EC 4.1.2.20). Themodified ED pathway involving non-phosphorylated intermediates was alsodiscovered in thermoacidophilic archaeon Thermoplasma acidophilum(Budgen et al. FEBS Lett. 196:207-210, 1986). The Thermoplasmaacidophilum metabolizes glyceraldehyde formed via thisnon-phosphorylated route by glyceraldehyde dehydrogenase to glycerate,which is phosphorylated to form 2-phosphoglycerate. This intermediate isthen converted to generate one molecule of pyruvate by enolase andpyruvate kinase. The non-phosphorylated ED pathway is a uniqueglycolysis pathway discovered only in the thermoacidophilic archaea, S.solfataricus and T. acidophilum. FIG. 1 is a non-phosphorylated EDpathway.

Another modified ED pathway involving phosphorylated intermediates isknown as a novel glycolysis route for glucose conversion to pyruvate insome species. This metabolism was first discovered by Szymona et al.from eubacteria Rhodobacter sphaeroides, and was also later found fromClostridia sp. and halobacteria (Conway, FEMS Microbiol. Rev. 103:1-28,1992). In this pathway, KDG produced by gluconate dehydratase isphosphorylated by KDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG)and is then cleaved by KDPG aldolase to pyruvate andglyceraldehyde-3-phosphate. The latter intermediate is oxidized topyruvate, a process that involves a conventional route, viaglyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase,enolase, and pyruvate kinase.

Gluconate dehydratase has described by Kersters et al., Antonie vanLeeuwenhoek. 37: 233-246 (1971); Kersters et al., Methods Enzymol. 42:301-304 (1975); Bender et al., Eur. J. Biochem. 40: 309-321 (1973);Bender et al., Methods Enzymol. 90: 283-287 (1982). The protein waspurified and characterized only from bacteria, Achromobacter species,and Clostridium pasteurianum, which metabolize gluconate via a formerglycolysis pathway. A comparison of the biochemical properties of eachenzymes shows that they are very different despite in vivo the samecatalytic reaction. In thermoacidophilic archaea, S. solfataricus, andT. acidophilum, however biochemical properties and detail mechanisms ofthe gluconate dehydratases are still unknown. Despite characterizationsof two enzymes from the above-described bacteria, no genes encodinggluconate dehydratase or partial amino acid sequences have beenreported. Hence, although recently the genomes of S. solfataricus and T.acidophilum were completely sequenced, putative genes encoding gluconatedehydratase could not be annotated in the database (She et al., Proc.Natl. Acad. Sci. USA. 98: 7835-7840, 2001; Ruepp et al., Nature407:508-513, 2000). In addition, the known gluconate dehydratases do notmaintain thermostability at temperatures greater than about 50° C. forprolonged periods up to several hours. Thus it is necessary to develop anovel gluconate dehydratase that can retain activity at hightemperatures for prolonged periods of time.

DISCLOSURE OF INVENTION Technical Problem

To solve the problems of the prior art, it is an aspect of the presentinvention to provide a novel thermostable gluconate dehydratase isolatedfrom thermoacidophilic archaea species.

It is another aspect of the present invention to provide an amino acidsequence of protein having gluconate dehydratase activity.

It is another aspect of the present invention to provide a nucleic acidsequence encoding a gluconate dehydratase.

It is another aspect of the present invention to provide a biologicalexpression system of a gluconate dehydratase and a transformantexpressing the gluconate dehydratase.

It is another aspect of the present invention to provide an in vitromethod of conversion aldonic acid into 2-keto-3-deoxy aldonic acid.

Technical Solution

In order to accomplish the aspects of the present invention, the presentinvention provides a polynucleotide encoding a gluconate dehydratase,wherein the gluconate dehydratase comprises a polynucleotide having atleast a 50% identity to a nucleic acid sequence encoding an polypeptidecomprising amino acid sequences of SEQ ID NO:2 or a polynucleotidecomplementary to the polynucleotide having at least a 50% identity to apolynucleotide encoding an polypeptide comprising amino acid sequencesof SEQ ID NO:2

The present invention provides a polypeptide comprising an amino acidsequence which is at least 50% identical to an amino acid sequence ofSEQ ID NO:2, wherein the polypeptide catalyzes dehydration of aldonicacid to 2-Keto-3-deoxy aldonic acid.

The present invention provides an expression construct comprising apolynucleotide comprising a nucleic acid sequence having at least a 50%identity to a nucleotide sequence encoding an polypeptide comprising anamino acid sequence of SEQ ID NO:2 or a polynucleotide complementary toa polynucleotide comprising a nucleic acid sequence having at least a50% identity to a nucleotide sequence encoding a polypeptide comprisingan amino acid sequence of SEQ ID NO: 2, wherein the polynucleotide isoperably linked to and under the regulatory control of a transcriptionand translation regulatory sequence.

The present invention provides an organism transformed with a vectorcomprising a polynucleotide encoding gluconate dehydratase, operablylinked to and under the regulatory control of a transcription andtranslation regulatory sequence.

The present invention provides a method for preparing a protein,comprising:

(a) preparing a vector comprising a polynucleotide encoding gluconatedehydratase, operably linked to and under the regulatory control of atranscription and translation regulatory sequence;

(b) introducing the vector into a host cell and selecting a transformantexpressing the protein;

(c) culturing the transformant under a condition which permits theprotein to be expressed; and

(d) purifying the protein from intracellular material of thetransformant, and wherein the protein catalyzes a dehydration of aldonicacid to 2-keto-3-deoxy aldonic acid.

The present invention provides a method of preparing an organismexpressing a protein, comprising:

(a) preparing a vector comprising a polynucleotide encoding gluconatedehydratase, operably linked to and under the regulatory control of atranscription and translation regulatory sequence;

(b) introducing the vector into a host cell; and

(c) selecting a transformant expressing the protein,

and wherein the protein catalyzes dehydration of aldonic acid to2-keto-3-deoxy aldonic acid.

The present invention provides a method of purifying gluconatedehydratase, comprising:

(a) harvesting a cell from the culture solution of gluconate dehydrataseproducing microorganism;

(b) obtaining a supernatant from intracellular material of the cell;

(c) conducting chromatography of the supernatant through a column packedwith DEAE-Sepharose to collect an eluant;

(d) conducting chromatography of the eluant of step (c) through a columnpacked with Q-Sepharose to collect an eluant;

(e) conducting chromatography of the eluant of step (d) through a columnpacked with Phenyl-Sepharose to collect an eluant; and

(e) conducting chromatography of the eluant of step (e) through a Mono QHR 5/5 column to collect a fraction.

The present invention provides a method for producing a 2-keto-3-deoxyaldonic acid from aldonic acid, comprising contacting the gluconatedehydratase to aldonic acid in water or an aqueous solvent attemperatures from 0° C. to 120° C. and pH 1.5 to 12, wherein the blendratio of gluconate dehydratase to aldonic acid is 1 ug: 0.01 to 1 mol.

DESCRIPTION OF DRAWINGS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings, wherein:

FIG. 1 is a non-phosphorylated ED pathway.

FIG. 2 is a vector map of pGNH.

FIG. 3 shows an effect of temperature on the activity of gluconatedehydratase from S. solfataricus.

FIG. 4 shows an effect of pH on gluconate dehydratase activity.

FIG. 5 is graph showing conversion result of 2-keo-3-deoxy gluconatefrom the gluconic acid when the Ss gluconate dehydratase was reacted toat pH 8.0 and 78° C. for 6 h

MODE FOR INVENTION

In the following detailed description, only selected embodiments of theinvention have been shown and described, simply by way of illustrationof the best mode contemplated by the inventors of carrying out theinvention. As will be realized, the invention may be modified in variousrespects, all without departing from the invention. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not restrictive.

As used herein, ‘purified’ or isolated’ refer to a nucleic acid orpolypeptide that is substantially free of cellular or viral materialwith which it is naturally associated, a culture median (when producedby recombinant DNA techniques), chemical precursors, or other chemicals(when chemically synthesized). Moreover, an isolated nucleic acidfragment is a nucleic acid fragment that is not naturally occurring as afragment and would not be found in the natural state.

As used herein, ‘nucleic acid or polynucleotide’ include both RNA andDNA, including genomic DNA, cDNA, and synthetic (e.g., chemicallysynthesized) DNA. Nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid orpolynucleotide can be a sense strand or an antisense strand. The nucleicacid or polynucleotide can be synthesized using oligonucleotide analogsor derivatives (e.g., inosine or phosphorothioate nucleotides).

As used herein, ‘thermostable’, when referring to an enzyme, means anenzyme which can function and is stable at high temperatures, is heatresistant, and will not denature at high temperatures.

A. Thermostable Gluconate Dehydratase

As used herein, the term ‘thermostable gluconate dehydratase’ in thecontext of the present invention refers to an enzyme which:

(1) is thermostable, i.e. substantially retains enzymatic activity uponexposure to heat at a temperature above 60-120° C., preferably above 80°C., and more preferably above 90° C.; and

(2) catalyzes aldonic acid to 2-keto-3-deoxy aldonic acid, and moreoverpreferably reacts gluconic acid to 2-keto-3-deoxy gluconic acid.

A gluconate dehydratase of the present invention can be isolated orpurified from the thermoacidophilic archaea species, preferablymicroorganisms belong to Sulfolobus genus, and more preferablySulfolobus solfataricus, Sulfolobus acidocaldarius, Sulfolobus shibatae,Sulfolobus tokodaii, Sulfolobus metallicus, Sulfolobus hakonensis,Sulfolobus brierleyi, Sulfolobus islandicus, Sulfolobus tengchongensis,Sulfolobus thuringiensis, Sulfolobus yangmingensis, Sulfolobus sp.,Thermoplasma acidophilum, Thermoplasma volcanium, Ferroplasmaacidophilum, or Sulfolobus strains AMP12/99, CH7/99, FF5/00, MV2/99,MVSoil3/SC2, NGB23/00, NGB6/00, NL8/00, NOB8H2, RC3, RC6/00, andRCS1/01.

The gluconate dehydratase of the present invention is thermostable andmaintains catalytic activity after a treatment of about 80° C. to about90° C. for 30 minutes. The thermostable range is from 0° C. to 120° C.,preferably from 20° C. to 100° C., and more preferably from 30° C. to90° C., and the optimum temperature is about 85° C. The gluconatedehydratase keeps its activity in a pH range of 1.5 to 12, preferablyfrom 1.5 to 10, more preferably from 4.0 to 9.0, and most preferablyfrom 6 to 8, affording a wide range of hybridization conditions in whichthe enzyme is active.

The aldonic acid as substrate for gluconate dehydratase may includeD-gluconate, D-Galactonate, D-Galactoheptonate, D-Arabonate,D-glucuronate, L-gulonate, D-tartarate, D-glucarate, L-isovalerate,L-threonate, D-ribonate, L-tartarate, D-gulonate, and D-galactarate butis not limited to. The embodiment of the present invention includes aD-gluconate as the preferred substrate for gluconate dehydratase derivedfrom S. solfataricus.

The gluconate dehydratase of the present invention includes apolypeptide with biological activity that is at least about 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence represented by SEQ ID NO:2. The nucleic acidsequence of the gluconate dehydratase includes a polynucleotide encodingpolypeptide that has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequencerepresented by SEQ ID NO:2 or its complements. The preferable nucleicacid sequence include a polynucleotide that is at least about 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to of SEQ ID NO:1 orits complements. The nucleic acid sequence can further contain animmediately contiguous sequence with both of the coding sequences (oneon the 5 end and one on the 3′ end).

In one embodiment, the gluconate dehydratase from S. solfataricus (Ss)that is designated herein as Ss gluconate dehydratase was isolated andcharacterized. The Ss gluconate dehydratase has about 320,000 to 380,000daltons as a native form, and has about 40,000 to 50,000 daltons asdetermined by SDS-PAGE under denaturing (reducing) conditions. Theseresults indicate that the S. solfataricus gluconate dehydratase in itsnative conformation is an octamer consisting of eight identicalsubunits. The sequence of gene coding by the Ss gluconate dehydrataseincludes the nucleotide sequence of SEQ ID NO:1.

B. Isolation and Purification of Thermostable Gluconate Dehydratase

The gluconate dehydratase can be isolated and purified fromthermoacidophilic archaea species, or chemically or biochemicallysynthesized by expression in a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plants, insects, and mammaliancells in culture).

The purification of gluconate dehydratase can be carried out by methodswell known to those skilled in the art, i.e., chromatography. Thechromatography can be conducted with the common resin attached thereto,with one or more kinds of functional groups selected from the groupconsisting of carboxy, carboxymethyl, sulpho, sulphomethyl,sulphoprophyl, aminoethyl, diethylaminoethyl, trimethyllaminomethyl,triethylaminoethyl, dimethyl-2-hydroxyethylaminomethyl,diethyl-2-hydroxypropylaminoethyl, phospho, alkyl (ex, hexyl-, octyl-,phenyl-) and hyroxylapatite. The matrix of the resin can be selectedfrom the group consisting of agarose, cellulose, dextran, polyacrylate,and polystyrene.

In one embodiment, the present invention provides a purification methodof gluconate dehydratase. The isolation and purification of gluconatedehydratase is performed at below room temperature to room temperature,preferably at about 4° C.

In the first step, the cells expressing the gluconate dehydratase areharvested, typically by centrifugation or filtration. In the steps, allbuffers contain a stabilizing agent or the like to increase the activityand yield of a gluconate dehydratase preparation.

In the second step, the cells are lysed and the supernatant issegregated and recovered from cellular debris. Lysis is typicallyaccomplished by mechanically applying physical stress and/or enzymaticdigestion, and segregation of the supernatant is usually accomplished bycentrifugation.

In the third step, the supernatant is further purified by chromatographywith a weak anionic exchange column. In the embodiment, the supernatantfrom the second step is applied to DEAE-Sepharose from Pharmacia(Piscataway, N.J., USA) equilibrated with a column buffer (50 mMtrihydroxymethylaminomethane (Tris), pH 7.2). The column is washed witha column buffer to remove unwanted macromolecules, and the bound proteinis then eluted off the column with the column buffer in a lineargradient of 0-1.0 molar (M) NaCl. In the case of Ss gluconatedehydratase, it is eluted at about 0.5 M NaCl. The eluant fractions arecollected and centrifuged to remove any insoluble material. Thecollected eluant is segregated, usually dialyzed, and then recovered toform a fraction containing partially purified gluconate dehydratase.

In the fourth step, the fraction containing gluconate dehydratase isfurther purified by chromatography with a strong anionic exchangecolumn. In the embodiment, the fraction is applied to Q-Sepharose fromPharmacia (Piscataway, N.J., USA) equilibrated with a column buffer (50mM trihydroxymethylaminomethane (Tris), pH 7.2). The column is washedwith the column buffer to remove unwanted macromolecules, and the boundprotein is then eluted off the column with the column buffer in a lineargradient of 0-1.0 molar (M) NaCl. In the case of Ss gluconatedehydratase, it is eluted at about 0.5 M NaCl. The eluant fractions arecollected and centrifuged to remove any insoluble material. Thecollected eluant is segregated, usually dialyzed, and then recovered toform a fraction containing partially purified Ss gluconate dehydratase.

For increasing purity of the gluconate dehydratase, the fractionprepared by the fourth step can be applied to a Phenyl-Sepharose columnequilibrated with 50 mM Tris-HCl, pH 7.2 containing 1.0 M NaCl. Afterwashing with the same buffer, the enzyme is eluted by a decreasing saltgradient of 1.0 to 0.0 M NaCl. Active fractions, collected at a flowrate of 0.5 ml/min, are pooled, concentrated by ultrafiltration, andloaded on a Mono Q HR 5/5 column equilibrated with 50 mM Tris-HCl, pH7.2. The enzyme is eluted with linear gradient of 0.0-1.0 M NaCl. Activefractions are collected, pooled, concentrated with an ultrafiltrationmembrane, and desalted with HiTrap™ desalting (Pharmacia, Sweden) toeliminate remaining NaCl in enzyme fractions.

C. Identification of the Isolated and Purified Gluconate Dehydratase andGene Thereof

The amino acid sequence of the isolated or/and purified gluconatedehydratase can be partially or fully determined by a method well knownin the art, such as by automated Edman degradation, and the like. Thedetermined amino acid sequence can be used for screening a novel proteinhaving homology in a database or/and for deducing coding nucleic acids.Then, a novel gene encoding gluconate dehydratase from various organismscan be screened through a suitable method such as PCR, sequencing, andso on.

The target organism may be an archaea species including Sulfolobussolfataricus, Sulfolobus acidocaldarius, Sulfolobus shibatae, Sulfolobustokodaii, Sulfolobus metallicus, Sulfolobus hakonensis, Sulfolobusbrierleyi, Sulfolobus islandicus, Sulfolobus tengchongensis, Sulfolobusthuringiensis, Sulfolobus yangmingensis, Sulfolobus sp., Thermoplasmaacidophilum, Thermoplasma volcanium, Ferroplasma acidophilum, andSulfolobus strains AMP12/99, CH7/99, FF5/00, MV2/99, MVSoil3/SC2,NGB23/00, NGB6/00, NL8/00, NOB8H2, RC3, RC6/00, and RCS1/01.

In one embodiment of the present invention, portions of the genomic DNAencoding at least six contiguous amino acids are synthesized and used asprobes to clone full-length genes of gluconate dehydratase. The nucleicacid encoding Ss gluconate dehydratase and a flanked sequence theretoare identified. The open reading frame for Ss gluconate dehydratase isshown in SEQ ID NO:1, and the nucleic acid sequence including the 3′ and5-flanked sequences is shown in SEQ ID NO:5.

Also, because there may not be a precisely exact match between thenucleotide sequence in the S. solfataricus as described herein and thatin the corresponding portion of the other species or strain, oligomerscontaining approximately 18 nucleotides (encoding the six amino acidstretch) may be necessary to obtain hybridization under conditions ofsufficient stringency to eliminate false positives.

Alternatively, polyclonal antiserum from rabbits immunized with purifiedSs gluconate dehydratase of the present invention can be used to probe aS. solfataricus partial genomic expression library to obtain theappropriate coding sequence.

D. Expression System of Thermostable Gluconate Dehydratase

A gluconate dehydratase can also be produced by recombinant DNA (rDNA)techniques. The gene encoding a thermostable gluconate dehydratase canbe operably linked to an expression system to form an rDNA capable ofexpression in a compatible host. Exemplary vectors and expression aredescribed herein.

The gene encoding a thermostable gluconate dehydratase includes a wildtype DNA or DNA altered by modification, substitution, deletion, oraddition of nucleic acid without substantially altering its catalyticactivity or thermostability, and such changes in sequence is acceptableand preferable where such changes impart desirable characteristics uponthe enzyme.

(1) Construction for Expression of Gluconate Dehydratase

For expression of the gluconate dehydratase, an expression constructincluding a polynucleotide encoding gluconate dehydratase, wherein thepolynucleotide is operably linked to and under the regulatory control ofa transcriptional and translational regulatory sequence, can beprepared. The transcriptional and translational regulatory sequences arethose which can function in a specific organism (i.e., bacteria, yeast,fungi, plants, insects, animals, and humans) cell or tissue to effectthe transcriptional and translational expression of the foreign genewith which they are associated and can be employed according to hostcell. The examples of transcriptional and translational regulatorysequences include a promoter, enhancer, polyadenylation signal, andterminator, but are not limited thereto.

The promoter can be derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operon encoding glycolytic enzymes such asglyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other promoters that have theadditional advantage of transcription controlled by growth conditionscan be employed, and examples are alcohol dehydrogenase 2, isocytochromeC, α-factor, acid phosphatase, heat shock proteins, degradative enzymesassociated with nitrogen metabolism, and enzymes responsible for maltoseor galactose utilization. And the promoter may be the known promotercontained in the common vectors lacI, lacZ, T3, T7, lamda P_(R), P_(L),trp, CMV immediate early, HSV thymidine kinase, early and late SV40,LTRs from retroviruses, and mouse metallothionein-I. Selection of theappropriate promoter is well within the level of ordinary skill in theart.

The enhancer is a cis-acting elements of DNA, usually from about 10 to1000 bp that act on a promoter to increase its transcription. Examplesinclude the SV40 enhancer on the last side of the replication origin bp100 to 270, a cytomegalovirus early promoter enhancer, the late side ofthe replication origin, and adenovirus enhancers.

The expression construct can further include a multi-cloning site,selectable marker, origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene and N-terminal identification peptide imparting desiredcharacteristics, e.g., a sequence for stabilizing or a simplifiedpurification process of expressed recombinant protein, a ribosomebinding site, or/and report gene. The expression construct may be acommon vector, and examples are a plasmid or viral vector. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample: pRSET, pTrcHis, pBAD, pTOPO, pTrxFus, pThioHis (Invitrogen),pET-19, 21, 24, 32, 43 (Novagen), pQE-30, -31, -32, pQE-40, -41, -42,pQE-50, -51, -52, pQE-16, -17, -18, pQE-60, pQE-70, pQE-9, -10, -11(Qiagen), pBluscript II (Stratagene), pTrc99a, pKK223-3, pDR540, pRIT2T(Amersham-Pharmacia), pXT1, pSG5 (Stratagene); pSVK3, pBPV, pMSG,pSVLS40 (Amersham-Pharmacia), pBR322 (ATCC37017); pKK223-3(Amersham-Pharmacia, Sweden), and pGEM1 (Promega, USA). However, anyother plasmid or vector may be used as long as they are replicable andviable in the host.

The suitable host for producing a recombinant protein includes aeukaryote, a prokaryote or virus. The eukaryote can be selected from thegroup consisting of a yeast, insect, animal, plant, and human, and acell derived therefrom, and the prokaryote can be a microorganismincluding E. coli, Streptomyces, Bacillus subtilis, and fungi. Examplesof the insect cell are Drosophila S2 and Spodoptera Sf9, Examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described by Gluzman (Cell, 23:175, 1981), and other celllines capable of expressing a compatible vector, for example, the C127,3T3, CHO, HeLa, and BHK cell lines.

(2) Establishment of Transformant

Techniques for generating transformants according to host cell type arewell known, for example calcium phosphate transfection, DEAE-Dextranmediated transfection, electroporation (Davis, L., Dibner, M, Battey,I., Basic Methods in Molecular Biology, 1986), and Agrobacteriumtumefaciens-mediated DNA transfer.

In one embodiment of the present invention, pGNH vectors harboring Ssgluconate dehydratase genes were prepared to be introduced intoEscherichia coli BL21(DE3) following select transformants. Thetransformants are designed as Escherichia coli BL21(DE3)/pGNH and beendeposited pursuant to Budapest Treaty requirements with the KoreanCollection for Type Cultures (KCTC), Taejon, Republic of Korea, in Apr.9. 2004, and were assigned accession number KCTC 10619BP.

The pGNH vector includes a Ss gluconate dehydratase coding portion andcontrol sequences at the 5 and 3′ termini of the coding portion onbetween BamHI and HindIII restriction sites. The sequence of pGNH isshown in SEQ ID NO:3, and loci of each component are represented inTable 1 and FIG. 2.

TABLE 1 pGNH vector Component Name loci Promoter T7 promoter 20-39Foreign gene gluD (gluconate dehydrates  208-1396 coding gene) Selectionmarker Ap (ampicillin resistance 2149-2963 gene) His-tag fusion region6xHis fusion region 100-207

(3) Production of the Recombinant Gluconate Dehydratase

Transformants are cultured in a condition for expressing the recombinantgluconate dehydratase according to the known method. The cultured cellsemployed in expression of proteins can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents, and such methods are wellknown to those skilled in the art. Cell are typically harvested bycentrifugation, disrupted by physical or chemical means, and theresulting crude extract retained for further purification.

In case of Escherichia coli BL21(DE3)/pGNH, a preferable culturecondition for expressing the recombinant Ss gluconate dehydrataseincludes follows:

Medium: Luria-bertani median, M9 medium, SOB (SOC) medium, TerrificBroth

-   -   Temperature: 20-40° C.    -   Culture time: 6-42 hrs

(4) Recover of Recombinant Protein

The recombinant gluconate dehydratase can be recovered and purified fromrecombinant cell cultures by any convenient method including ammoniumsulfate precipitation, acetone precipitation, acid extraction, anionexchange chromatography, cation exchange chromatography, hydrophobicinteraction chromatography, phospho-cellulose chromatography, affinitychromatography, hydroxylapatite chromatography, and lectinchromatography, and preferably by a method of the present inventionmentioned above. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

Depending upon the host employed in a recombinant production procedure,the recombinant Ss gluconate dehydratase of the present invention may ormay not be a post-translational modification, such as throughglycosylation, phosphorylation, and acetylation. Enzymes of theinvention also may or may not include an initial methionine amino acidresidue.

In an embodiment of the present invention, recombinant Ss gluconatedehydratase from Escherichia coli BL21(DE3)/pGNH is purified by nickelaffinity chromatography.

E. Use of Gluconate Dehydratase

The gluconate dehydratase may be employed for any purpose in which suchenzyme activity is necessary or desired. In a preferred embodiment theenzyme is employed for catalyzing the dehydration of aldonic acid. Thedehydration of aldonic acid may be used for the production ofcarbohydrate intermediates used in pharmaceutical, agricultural, andother chemical products.

The gluconate dehydratase, their fragments, derivatives, or analogiesthereof, or recombinant gluconate dehydratase, can be used as animmunogen to produce antibodies thereto. These antibodies can be, forexample, polyclonal or monoclonal antibodies. The present invention alsoincludes chimeric, single chain, and humanized antibodies, as well asFab fragments, and the product of a Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

Antibodies generated against the gluconate dehydratase can be obtainedby direct injection of the enzymes into an animal or by administeringthe enzymes to an animal, preferably a nonhuman. The antibody obtainedthen binds the gluconate dehydratase itself. In this manner, even asequence encoding only a fragment of the gluconate dehydratase can usedto generate antibodies and can then be used to isolate the enzyme fromcells expressing that gluconate dehydratase.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today, 4:72, 1983), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., In Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., pp77-96, 1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodiesfor immunogenic enzyme products of the present invention. Also,transgenic mice may be used to express humanized antibodies toimmunogenic enzyme products of this invention.

Antibodies generated against the gluconate dehydratase of the presentinvention may be used in screening for similar enzymes from otherorganisms and samples. Antibodies may also be employed as a probe toscreen gene libraries generated from this or other organisms to identifythis or cross reactive activities.

F. Production of 2-keto-3-deoxy Aldonic Acids from Aldonic Acids

The gluconate dehydratase dehydrates aldonic acid to 2-keto-3-deoxyaldonic acid. Thus the gluconate dehydratase of the present inventioncan be used for production 2-keto-3-deoxy aldonic acid from aldonicacid.

The present invention provides a method of producing 2-keto-3-deoxyaldonic acid from aldonic acid including contacting the gluconatedehydratase to aldonic acid in water or an aqueous solvent attemperatures from 0° C. to 120° C. and pH 1.5 to 12, wherein the blendratio of gluconate dehydratase to aldonic acid is 1 ug: 0.01 to 1 mol.

The gluconate dehydratase can be selected from the group consisting ofan isolated native gluconate dehydratase, a chemically synthesizedgluconate dehydratase, a recombinant gluconate dehydratase, andderivatives thereto.

The aldonic acid prefers D-gluconate, D-Galactonate, D-Galactoheptonate,D-Arabonate, D-glucuronate, L-gulonate, D-tartarate, D-glucarate,L-isovalerate, L-threonate, D-ribonate, L-tartarate, D-gulonate, andD-galactarate.

The dehydration reaction of aldonic acid is conveniently carried out attemperatures from 0° C. to 120° C., preferably from 20° C. to 100° C.,and most preferably from 30° C. to 90° C.

The suitable pH for effecting the enzyme reaction is from 1.5 to 12,preferably from 1.5 to 10, and most preferably from 4.0 to 9.0.

The concentration of the substrate and aldonic acids in the reactionmixture is conveniently from 1 to 700 g/L, preferably from 10 to 500g/L, and most preferably from 50 to 200 g/L.

The optimum condition for the dehydration reaction of aldonic acidincludes the gluconate dehydratase concentration of 0.1-1 mg/mL,substrate concentration of 100-200 mM, reaction time of less than 6 hr,temperature of 70-95° C., and pH of 7.0-8.0.

The reaction is conveniently carried out in water or an organic solvent.The organic solvent is selected from the group consisting of alcohol,0.01 to 100% of aqueous alcohol, and a mixture of several alcohols,aromatic hydrocarbon, and aliphatic hydrocarbon. The alcohol ispreferably a C₁₋₆-alkanol, such as methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, or tert-butanol. The aliphatichydrocarbon alcohol is preferably heptane or isooctane, and the aromatichydrocarbon alcohol is preferably benzene or toluene. From an economicand environmental point of view, as little organic solvent as possibleis used in the industrial process.

The dehydration reaction can be carried out in a condition of additionof an antioxidant, such as 2-mercaptoethanol, dithiothreitol, orcysteine, to prevent the degradation of the produced 2-keto-3-deoxy acidanalogies.

As an alternative to a gluconate dehydratase itself, the reactionmixture may comprise an organism having gluconate dehydratase activity.

For the reaction, any form of the gluconate dehydratase enzyme can beused, in particular an enzyme solution, the immobilized enzyme, intactcells of the organism having gluconate dehydratase activity, andimmobilized cells having gluconate dehydratase activity.

The following examples are provided to further illustrate the presentinvention and are not intended to limit the invention beyond thelimitations set in the appended claims.

Example 1 Cultivating sulfolobus solfataricus and Preparing Ss CellPaste

The following describes how the hyperthermophilic archaeon S.solfataricus is routinely grown in a 3.7 liter fermentor for the purposeof obtaining cell mass in sufficient quantities for large scale proteinpurification.

For culture maintenance, S. solfataricus P2 (DSM1617) is routinely grownat 75-85° C. as a closed shaking culture at a volume of 100 ml. Theorganism was cultivated in the medium (per liter, 3.0 g glucose, 3.0 gyeast extract, 1.3 g (NH₄)₂SO₄, 0.28 g KH₂PO₄, 0.25 g MgSO₄.7H₂O, 0.07 gCaCl₂.H₂O) containing 1 ml trace metal solution (20 mg FeCl₃.H₂O, 4.5 mgNa₂B₄O₇.H₂O, 1.8 mg MnCl₂.H₂O, 0.05 mg ZnSO₄.H₂O, 0.05 mg CuCl₂.H₂O,0.04 mg VOSO₄.H₂O, 0.03 mg Na₂MoO₄.H₂O, 0.01 mg CoSO₄.H₂O per liter).The final pH was adjusted to pH 3.0 with 1 M H₂SO₄. Cultures were grownaerobically in a 3.7-liter fermentor (KLF 2000, Bioengineering AG,Switzerland) at 78° C. while being stirred at 400 rpm. Growth wasmonitored spectrophotometrically at 540 nm.

Example 2 Purification of Gluconate Dehydratase from sulfolobussolfataricus

Cells of S. solfataricus (frozen wet cell weight 35 g) were harvested bycentrifugation (5000×g, 30 min, 4° C.) and washed twice with 50 mMTris-HCl (pH 7.2). Cell pellets were re-suspended in 50 mM Tris-HCl (pH7.2), and disrupted by sonication for 1 h at 50% output. Crude extractswere heated at 90° C. for 20 min., and heat-denatured proteins and celldebris were removed by centrifugation (50000×g, 1 h, 4° C.). To thesupernatant solution was added solid (NH₄)₂SO₄ up to 40% saturation torecover a fraction containing the activity of gluconate dehydratase.After centrifugation (50000×g, 1 h, 4° C.), the soluble fraction wasdialyzed in 50 mM Tris-HCl (pH 7.2). The homogenate was loaded onto aDEAE-Sepharose column (2.5×16 cm) previously equilibrated with 50 mMTris-HCl, pH 7.2, and the elution was performed with a three bed volumeof the same buffer, followed by a linear gradient of 0.0-1.0 M NaCl.Fractions (5 ml each) were collected at a flow rate of 1 ml/min. Thosewith gluconate dehydratase activity were pooled, concentrated byultrafiltration on a Vivaspin™ concentrator membrane (Vivascience,Lincoln, UK) and loaded on a Phenyl-Sepharose column (1.0×10 cm)equilibrated with 50 mM Tris-HCl, pH 7.2, containing 1.0 M NaCl. Afterwashing with the same buffer, the enzyme was eluted by a decreasing saltgradient of 1.0 to 0.0 M NaCl. Active fractions, collected at a flowrate of 0.5 ml/min, were pooled, concentrated by ultrafiltration, andloaded on a Mono Q HR 5/5 column (0.5×5 cm) equilibrated with 50 mMTris-HCl, pH 7.2. The enzyme was eluted with a linear gradient of0.0-1.0 M NaCl. Active fractions, collected at a flow rate of 0.5ml/min, were pooled, concentrated with ultrafiltration membrane, anddesalted with HiTrap™ desalting (Pharmacia, Sweden) to eliminateremaining NaCl in enzyme fractions.

The resulting product is referred to as Ss gluconate dehydratase. Theresultant Ss gluconate dehydratase was determined to be 95% homogeneousby analysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Example 3 Assay of the Gluconate Dehydratase

Ss Gluconate dehydratase activity was measured by the semicarbazidemethod or TBA (thiobarbituric acid) assay.

The semicarbazide method was performed as follows: an enzyme reaction ofa total volume a 400 μl was incubated at 78° C. in 50 mM Tris-HClbuffer, pH 7.0, with 10 mM gluconate and an enzyme solution. After 30min, the enzyme reaction was stopped by the addition of 100 μl 2.0 MHCl. To this solution, 300 μl of semicarbazide solution (1.0% (w/v)semicarbazide hydrochloride and 1.5% (w/v) sodium acetate dissolved indistilled water) was added and incubated at 30° C. for 15 min. The finalreaction mixture was diluted with 500 μl distilled water and thenmeasured at 250 nm. The absorbance coefficient of the semicarbazoneformation toward 2-keto-3-deoxy gluconate (KDG) was taken to be0.571×10³ M⁻¹ cm⁻¹.

TBA assay was performed as follows: the reaction mixtures of 50 μl wereoxidized by 125 μl of 25 mM periodic acid in 0.25 M H₂SO₄ at roomtemperature for 20 min. To terminate oxidation, 250 μl of 2% (w/v)sodium arsenite dissolved in 0.5 M HCl was added to the reactants.Finally, after adding 1 ml of 0.3% TBA to the reactants, the reactionmixtures was heated at 100° C. for 10 min. Produced red chromophore wasmonitored at 549 nm after adding an equal volume of DMSO. The absorbancecoefficient of thiobarbituric acid chromophore toward KDG was estimatedto be 0.347×10³ M⁻¹ cm⁻¹. One unit of gluconate dehydratase was theamount of the enzyme producing 1 μmol of 2-keto-3-deoxy gluconate permin. from gluconate under this assay conditions. All enzyme activitieswere determined in three plicate.

Example 4 Identification of the Gene Encoding Gluconate DehydrataseThrough N-Terminus Sequencing

To analyze N-terminal sequencing, purified protein was loaded on anSDS-PAGE blotted onto a PVDF membrane, and excised. The N-terminalsequence of gluconate dehydratase purified from S. solfataricus wasdetermined by Edman degradation to be MRIREIEPIV. The deduced amino acidsequence (SEQ ID NO: 2) of gluconate dehydratase was exactly inagreement with SSO3198, which coded for the 45-kDa protein in the S.solfaricus P2 genome database. The predicted protein size in the genomicdatabase corresponded to the single band of purified enzyme in thedenaturing gel. Consequently, this purified protein is gluconatedehydratase, and the ORF annotated by SSO3198 is the gene, which wasnamed gnh, encoding gluconate de hydratase in S. solfataricus.

Example 5 Characterizing Ss Gluconate Dehydratase

5-1. Substrate Specificities

For analysis of substrate specificities of gluconate dehydrates, a 10 mMsolution of each aldonic acid containing carbon chains ranging from C₄to C₇ were incubated together with 40 μg/mL of purified protein. Theamount of product formation was measured by the semicarbazide method,which showed 100% conversion for D-gluconate after incubation under thestandard condition. Substrate specificity of gluconate dehydratase forsugar acids was determined by the method measuring 2-keto-3-deoxyanalogues yielded from aldonic acids. Sugar acids tested are as follows:D-gluconate, D-galactonate, D-galactoheptonate, D,L-arabonate,D-glucuronate, D,L-gulonate, D,L-tartarate, D-glucarate,D,L-isovalerate, L-threonate, D-ribonate, D-galactarate, D-xylonate,D-galacturonate, D-glucitol, D-mannonate, and D,L-glycerate. Kineticparameters for gluconate dehydratase were determined using D-gluconate(0.1 to 40 mM). All experiments were performed in three plicate.

The results of Ss gluconate dehydratase activity for the aldonic acidsare shown in Table 2. The Ss gluconate dehydratase showed higherselectivity to D-gluconate than any other adonic acids. D-Galactonateand D-galactoheptonate could be used as substrates for the enzyme.Negligible but detectable activities (less than 1% of activity towardD-gluconate) were observed for the following substrates: D-glucuronate,L-gulonate, D-tartarate, D-glucarate, Et L-isovalerate, L-threonate,D-ribonate, L-tartarate, D-gulonate, and D-galactarate. It thereforeappears that the enzyme has a preference to D-gluconate.

TABLE 2 Relative Probable structure of Substrates activity (%)dehydration products D-Gluconate 100.0 2-keto-3-deoxy-D-gluconateD-Galactonate 2.8 2-keto-3-deoxy-D-galactonate D-Galactoheptonate 1.62-keto-3-deoxy-D-galactoheptonate Substrates Relative Probable structureof dehydration activity (%) products D-Arabonate 0.72-keto-4,5-dihydroxy-D-valeric acid Less than 1% activity on thefollowing substrates; D-glucuronate (0.65), L-gulonate (0.41),D-tartarate (0.41), D-glucarate (0.32) D,L-isovalerate (0.25),L-threonate (0.16), D-ribonate (0.16), L-tartarate (0.16), D-gulonate(0.16), and D-galactarate (0.10). No reaction on the followingsubstrates: L-arabonate, D-xylonate, D-galacturonate, D-glucitol,D-mannonate, and D,L-glycerate. The relative enzyme activity was assayedby measuring the 2-keto-3-deoxy analogues produced from 10 mM each ofaldonic acid containing 1 mM CoCl2 in 50 mM Tri-HCl buffer (pH 7.0) for30 min at 78?C. using the semicarbazide method.

Biochemical and kinetic parameters for the enzyme were determined usingthe assay method described above under standard conditions.

5-2. Kinetic Parameters

Values for V_(max) and K_(m) were determined from Lineweaver bulk plots.The rate dependence on substrate concentration followed Michaelis-Metenkinetics. From Lineweaver-Burk plots, K_(m) and V_(m) values of 16.7 mMand 34.5 units/mg were determined with D-gluconate as the substrate. Theturnover number (k_(cat)) was cat calculated as 333 s⁻¹ for gluconatedehydratase, and the value of k_(cat)/K_(m) was 19.9.

5-3. Optimum Temperature

The temperature profile for enzyme activity was determined between 40and 100° C. FIG. 3 shows an effect of temperature on the activity ofgluconate dehydratase from S. solfataricus. The purified gluconatedehydratase displayed optimal activity between 80 and 90° C. Enzymeactivity was not detectable below 60° C.

5-4. Thermostability

Enzyme thermostability was determined at 80, 90, and 100° C. byincubating enzyme solution (50 μg/ml) in 50 mM Tris-HCl (pH 7.2). At anappropriate time, samples were taken and completely cooled on ice andthen measured for residual activities under standard conditions. Thethermostability of purified gluconate dehydrates was measured at 80, 90,and 100° C. At 80° C., the optimal temperature for growth of S.solfataricus P2, the gluconate dehydratase was very stable over 2 hours.At 90° C., enzyme activity decreased below 50% after a 2 hourincubation. At 100° C., however, the enzyme had a half-life of less than40 min.

5-5. Optimum pH

The effect of pH on gluconate dehydratase activity was determined at 78°C. in a citric acid-NaOH buffer (pH 2.7-5.0), 50 mM Tris-HCl buffer (pH5.8-8.0), and 50 mM glycine-NaOH buffer (pH 8.5-10.5).

FIG. 4 shows an effect of pH on gluconate dehydratase activity; 50 mMcitric acid-NaOH (▪), 50 mM Tris-HCl (), and 50 mM glycine-NaOH buffer(◯). In FIG. 4, within the pH range from pH 2.7 to pH 10.5, the activityof purified enzyme displayed an optimum between pH 7.0 to 8.0.

Example 6 Mass Determination of Native Gluconate Dehydratase

Pure Ss gluconate dehydratase (100 μg) of EXAMPLE 2 was chromatogramedthrough a Sephacryl S-200 column (1.0×89 cm) using the gel filtrationcalibration kit (Pharmacia Biotech, Sweden). The equilibrium and elutionbuffer used was 50 mM Tris-HCl, pH 7.2, containing 150 mM NaCl, and theflow rate was 0.5 ml/min. The molecular weight markers used werethyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa),aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), chymotrypsin (25kDa), and ribonuclease A (13.7 kDa). Proteins were detected at 280 nm,and gluconate dehydratase activity was measured by the standard method.The gluconate dehydratase molecular weight was calculated byinterpolation on a plot of log molecular mass against the K_(av) valuesfollowing the recommended procedure.

The native molecular weight of purified enzyme was 357±42 kDa, asmeasured on a calibrated Sephacryl S-200 column with standard molecularweight markers. The molecular mass of denaturated gluconate dehydratasedetermined from SDS-PAGE was approximately 44 kDa. These resultsindicate that the S. solfataricus gluconate dehydratase in its nativeconformation is an octamer consisting of eight identical subunits.

Example 7 Cloning of Gene Coding Gluconate Dehydratase from Sulfolobussolfataricus

7-1. Cloning

The gene coding thermostable gluconate dehydratase was cloned from thehyperthermophilic archaeon Sulfolobus solfataricus (Ss).

Amino terminal protein microsequencing was performed by the Korea BasicScience Institute (KBSI) (Daejeon, Korea) on 100 picomoles (pmol) ofhomogeneous native Ss gluconate dehydratase prepared as described inExample 2. The sequence of the 10 N-terminal amino acid residues therebyobtained was later shown to correspond exactly with deduced residuesshown in SEQ ID NO 4 from residue 1 to residue 10.

DNA encoding the Ss gluconate dehydratase of the present invention, SEQID NO 1, was initially amplified from Sulfolobus solfataricus genomicDNA by the PCR technique using the primer set of SEQ ID NO:5 and 6,including the BamHI restriction site and HindIII restriction site. Theamplified fragments were inserted into the BamHI and HindIII sites ofpGEM-T easy (Promega, USA) and the resulting vector was digested by eachBamHI and HindIII restriction enzyme. The 1,188 bp fragments wereligated into the BamHI and HindIII sites of pRSET vector (Invitrogen,USA) including antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), and IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag, and restriction enzyme sites,and the resulting vector was designated as pGNH. The pGNH contains thecomplete 3,993 bp fragment encoding Ss gluconate dehydratase flanked atthe fragment's termini by BamHI and HindIII.

The pGNH was then used to transform the E. coli strain BL21(DE3) whichis a protease-deficient mutant to protect heterologously expressedproteins against protease. Transformants were selected by growing in LBmedium supplemented with ampicillin, and were harvested to confirmedwhether the gnh gene was placed therein by restriction analysis.

7-2. Expression

Transformants were grown overnight in a liquid culture in LB mediasupplemented with Amp (100 μg/ml). The overnight culture was used toinoculate a large culture at a ratio of 1:100 to 1:250. The cells weregrown to an optical density (OD₆₀₀) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to a finalconcentration of 1 mM IPTG induces by inactivating the lac repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 4 to 6 hours, and were then harvested by centrifugation.

Example 8 Purification of Recombinant Ss Gluconate Dehydratase

Recombinant Ss gluconate dehydrates was purified from E. coli containingthe plasmid pGNH described in Example 7.

Cultures of Escherichia coli BL21(DE3)/pGNH were prepared as before, and30 grams of cultured cells were isolated, admixed in 120 ml lysis buffer(prepared as in Example 2), and sonicated 10 times for 6 minutes each atfull power. The resulting lysate was centrifuged for 30 minutes at 7,000rpm. The supernatant from centrifugation was isolated and then placedfor 20 minutes in a 90° C. water bath. The heat-denaturated solution wasthen centrifuged as above and the resultant was isolated and then loadedon an IMAC^(?) column equilibrated in 50 mM Tris-HCl, pH 7.2 asdescribed in Example 2. The column was washed with 3 column volumes ofthe same buffer, and then eluted with a gradient of 0-0.2 M imidazole inthe same buffer, thereby collecting gradient elution fractions. Thegluconate dehydratase activity assay was performed on each fraction, andpeak activity fractions were pooled and dialyzed in 50 mM Tris-HCl (pH7.2).

Following dialysis, the dialysate was loaded on a Q-Sepharose columnequilibrated with 50 mM Tris-HCl, pH 7.2, as described in Example 2. Thecolumn was washed with 3 column volumes and eluted with a 0-1.0 M NaClgradient in 50 mM Tris-HCl (pH 7.2). Peak activity fractions were pooledand assayed, and active fractions were pooled and concentrated 10-20fold in a Vivaspin™ concentrator (Vivascience, Lincoln, UK). Theconcentrated pool was then dialyzed against a final dialysis buffer toform purified recombinant Ss gluconate dehydratase.

The activity of the recombinant Ss gluconate dehydratase was determinedby the method described in Example 3.

Example 9 Dehydration of Gluconic Acid to 2-Keto-3-deoxygluconate byRecombinant Gluconate Dehydratase

The recombinant gluconate dehydratase from S. solfaricus was used forthe dehydration of gluconic acid to 2-keto-3-deoxy gluconate.

The reaction mixture consisted of 1, 5, 10, 50, and 100 mM gluconic acidsodium salt (Sigma Chemical Co., St. Louis, Mo., USA), and the Ssgluconate dehydratase in 50 mM Tris-HCl buffer (pH 8.0). The gluconatedehydratase was added at a concentration of 3.5 mg/ml, and the reactionwas carried out at 78° C. for 6 hours. 2-Keto-3-deoxy gluconate wasassayed by the standard procedure described in Example 3. 2-Keto-3-deoxygluconate was produced by the Ss gluconate dehydratase as shown in FIG.5.

The optimum conditions of recombinant Ss gluconate dehydratase fordehydrating aldonic acid to 2-keto-3-deoxy aldonic acid follow:

Enzyme concentration: 0.1-1 mg/mL

Substrate concentration: 100-200 mM

Reaction time: within 6 hours

Temperature: about 80° C.

pH: 7.5-8.0

Sequence List Text

SEQ ID NO:1—Open Reading Frame encoding gluconate dehydratase fromSulfolobus solfataricus.

SEQ ID NO:2—amino acid sequence of gluconate dehydratase from Sulfolobussolfataricus.

SEQ ID NO:3—nucleic acid sequence of pGNH vector.

SEQ ID NO:4—N-terminal amino acid sequence from the gluconatedehydratase purified from Sulfolobus solfataricus.

SEQ ID NO:5—nucleic acid sequence of sense primer with BamHI restrictionsite.

SEQ ID NO:6—nucleic acid sequence of antisense primer with HindIIIrestriction site.

1. An isolated or purified polynucleotide encoding a gluconatedehydratase, wherein the gluconate dehydratase comprises apolynucleotide having at least a 50% identity to a nucleic acid sequenceencoding a polypeptide comprising amino acid sequences of SEQ ID NO:2,or a polynucleotide complementary to the polynucleotide having at leasta 50% identity to a polynucleotide encoding an polypeptide comprisingamino acid sequences of SEQ ID NO:2.
 2. The polynucleotide according toclaim 1, wherein the polynucleotide is DNA.
 3. The polynucleotideaccording to claim 1, wherein the polynucleotide is RNA.
 4. Thepolynucleotide according to claim 1, wherein the polynucleotidecomprises nucleotide sequences of SEQ ID NO:1
 5. A polypeptidecomprising an amino acid sequence which is at least 50% identical toamino acid sequences of SEQ ID NO:2, wherein the polypeptide catalyzesdehydration of aldonic acid to 2-Keto-3-deoxy aldonic acid.
 6. Anexpression construct comprising the polynucleotide of claim 2, whereinthe polynucleotide is operably linked to and under the regulatorycontrol of a transcription and translation regulatory sequence.
 7. Anorganism transformed with an expression construct according to claim 6.8. The organism according to claim 7, wherein the organism is selectedfrom the group consisting of a prokaryote, a eukaryotic cell, and a cellderived thereof.
 9. The organism according to claim 7, wherein theorganism is Escherichia coli BL21(DE3)/pGNH (KCTC10619BP).
 10. A methodfor preparing a protein, comprising: (a) preparing a vector comprising apolynucleotide of claim 1, operably linked to and under the regulatorycontrol of a transcription and translation regulatory sequence; (b)introducing the vector into a host cell and selecting a transformantexpressing the protein; (c) culturing the transformant under a conditionwhich permits the protein to be expressed; and (d) purifying the proteinfrom the cultures, wherein the protein catalyzes a dehydration ofaldonic acid to 2-keto-3-deoxy aldonic acid.
 11. A method of preparingan organism expressing a protein, comprising: (a) preparing a vectorcomprising a polynucleotide of claim 1, operably linked to and under theregulatory control of a transcription and translation regulatorysequence; (b) introducing the vector into a host cell; and (c) selectinga transformant expressing the protein, wherein the protein catalyzesdehydration of aldonic acid to 2-keto-3-deoxy aldonic acid.
 12. A methodof purifying a gluconate dehydratase, comprising: conductingchromatography of a culture solution or a cell from a gluconatedehydratase producing microorganism through a column packed with resinattached to one or more kinds of functional groups selected from thegroup consisting of carboxy, carboxymethyl, sulpho, sulphomethyl,sulphoprophyl, aminoethyl, diethylaminoethyl, trimethylaminomethyl,triethylaminoethyl, dimethyl-2-hydroxyethylaminomethyl,diethyl-2-hydroxypropylaminoethyl, phospho, alkyl and hydroxylapatite,and wherein the matrix of the resin is selected from the groupconsisting of agarose, cellulose, dextran, polyacrylate, andpolystyrene.
 13. The method according to claim 12, wherein themicroorganism is thermoacidophilic archaea species.
 14. The methodaccording to claim 12, wherein the microorganism belongs to Sulfolobusgenus.
 15. The method according to claim 12, wherein the microorganismis selected from the group consisting of Sulfolobus solfataricus,Sulfolobus acidocaldarius, Sulfolobus shibatae, Sulfolobus tokodaii,Sulfolobus metallicus, Sulfolobus hakonensis, Sulfolobus brierleyi,Sulfolobus islandicus, Sulfolobus tengchongensis, Sulfolobusthuringiensis, Sulfolobus yangmingensis, Sulfolobus sp., Thermoplasmaacidophilum, Thermoplasma volcanium, Ferroplasma acidophilum, andSulfolobus strains AMP12/99, CH7/99, FF5/00, MV2/99, MVSoil3/SC2,NGB23/00, NGB6/00, NL8/00, NOB8H2, RC3, RC6/00, or RCS1/01.
 16. A methodfor producing a 2-keto-3-deoxy aldonic acid from aldonic acid,comprising: contacting the gluconate dehydratase to aldonic acid inwater or an aqueous solvent at a temperature from 0° C. to 120° C. and apH of 1.5 to 12, wherein the blend ratio of a gluconate dehydratase toaldonic acid is 1 ug: 0.01 to 1 mol.
 17. The method according to claim16, wherein the gluconate dehydratase is selected from the groupconsisting of an isolated native gluconate dehydratase, a chemicallysynthesized gluconate dehydratase, a recombinant gluconate dehydratase,and derivatives thereto.
 18. The method according to claim 16, whereinthe aldonic acid is selected from the group consisting of D-gluconate,D-Galactonate, D-Galactoheptonate, D-Arabonate, D-glucuronate,L-gulonate, D-tartarate, D-glucarate, L-isovalerate, L-threonate,D-ribonate, L-tartarate, D-gulonate, and D-galactarate.